Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Grk7 but not Grk1 undergoes cAMP-dependent phosphorylation in zebrafish cone photoreceptors and mediates cone photoresponse recovery to elevated cAMP.

In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases (GRKs) is essential for sustained visual function. Previous in vitro analysis demonstrated that GRK1 and GRK7 are phosphorylated by PKA, resulting in a reduced capacity to phosphorylate rhodopsin. In vivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones in vivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation in vivo. Lastly, immunoblot analyses of rod grk1a-/- and cone grk1b-/- zebrafish and Nrl-/- mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation in vivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.

Broad spectrum metabolomics for detection of abnormal metabolic pathways in a mouse model for retinitis pigmentosa.

Retinitis pigmentosa (RP) is a degenerative disease of the retina that affects approximately 1 million people worldwide. There are multiple genetic causes of this disease, for which, at present, there are no effective therapeutic strategies. In the present report, we utilized broad spectrum metabolomics to identify perturbations in the metabolism of the rd10 mouse, a genetic model for RP that contains a mutation in Pde6ß. These data provide novel insights into mechanisms that are potentially critical for retinal degeneration. C57BL/6J and rd10 mice were raised in cyclic light followed by either light or dark adaptation at postnatal day (P) 18, an early stage in the degeneration process. Mice raised entirely in the dark until P18 were also evaluated. After euthanasia, retinas were removed and extracted for analysis by ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-QTOF-MS). Compared to wild type mice, rd10 mice raised in cyclic light or in complete darkness demonstrate significant alterations in retinal pyrimidine and purine nucleotide metabolism, potentially disrupting deoxynucleotide pools necessary for mitochondrial DNA replication. Other metabolites that demonstrate significant increases are the Coenzyme A intermediate, 4'-phosphopantothenate, and acylcarnitines. The changes in these metabolites, identified for the first time in a model of RP, are highly likely to disrupt normal energy metabolism. High levels of nitrosoproline were also detected in rd10 retinas relative to those from wild type mice. These results suggest that nitrosative stress may be involved in retinal degeneration in this mouse model.

Reduced phosphoCREB in Müller glia during retinal degeneration in rd10 mice.

PURPOSE: The mechanisms that trigger retinal degeneration are not well understood, despite the availability of several animal models with different mutations. In the present report, the rd10 mouse, a model for retinitis pigmentosa (RP) that contains a mutation in the gene for PDE6ß (Pde6b), is used to evaluate gliosis, as a marker for retinal stress, and cyclic AMP response element binding protein (CREB) phosphorylation, which may be important early in retinal degeneration. METHODS: Wild-type C57Bl6J and rd10 mice raised under cyclic light were examined for changes in gliosis and CREB phosphorylation for approximately 3 weeks beginning at P14 to P17 using immunocytochemistry. Mice raised under normal cyclic light and in complete darkness were also compared for changes in CREB phosphorylation. RESULTS: Gliosis in rd10 mice raised under cyclic light was apparent at P17, before extensive degeneration of the photoreceptor layer is visible, and increased over time. Phosphorylation of CREB at Ser133 (pCREB) was detected in Müller glia (MG) in the wild-type and rd10 mice. However, at all phases of photoreceptor degeneration, the pCREB levels were lower in the rd10 mice. We also observed extensive migration of MG cell bodies to the outer nuclear layer (ONL) during degeneration. In contrast to the mice raised under cyclic light, the rd10 mice raised in the dark exhibited slower rates of degeneration. When the dark-reared mice were exposed to cyclic light, the photoreceptor layer degenerated within 4 days to approximately one to two rows of nuclei. Interestingly, the pCREB levels in the MG also decreased during this 4-day cyclic light exposure compared to the levels in the rd10 mice raised continuously in the dark. CONCLUSIONS: The results of these studies suggest that photoreceptors communicate directly or indirectly with MG at early stages, inducing gliosis before extensive retinal degeneration is apparent in rd10 mice. Surprisingly, phosphorylation of CREB is downregulated in the MG. These results raise the interesting possibility that Müller glia undergo CREB-mediated transcriptional changes that influence photoreceptor degeneration either positively or negatively. Unlike canine models of RP, no increase in pCREB was observed in photoreceptor cells during this period suggesting possible mechanistic differences in the role of CREB in photoreceptors between these species.

Accumulation patterns of tobacco root allelopathicals across different cropping durations and their correlation with continuous cropping challenges.

Introduction: Continuous cropping challenges have gradually emerged as pivotal factors limiting the sustainable development of agricultural production. Allelopathicals are considered to be the primary obstacles. However, there is limited information on allelopathic accumulation across various continuous cropping years and its correlation with the associated challenges. Methods: Tobacco was subjected to varying planting durations: 1 year (CR), 5 years (CC5), 10 years (CC10), and 15 years (CC15). Results: Our findings unveiled discernible disparities in tobacco growth patterns across diverse continuous cropping periods. Notably, the most pronounced challenges were observed in the CC5 category, characterized by yield reduction, tobacco black shank outbreaks, and a decline in beneficial flora. Conversely, CC15 exhibited a substantial reduction in challenges as the continuous cropping persisted with no significant differences when compared to CR. Within the tobacco rhizosphere, we identified 14 distinct allelopathic compounds, with 10 of these compounds displaying noteworthy variations among the four treatments. Redundancy analysis (RDA) revealed that eight allelopathic compounds exhibited autotoxic effects on tobacco growth, with MA, heptadecanoic acid, and VA ranking as the most potent inhibitors. Interaction network highlighted the pivotal roles of VA and EA in promoting pathogen proliferation and impeding the enrichment of 13 beneficial bacterial genera. Furthermore, a structural equation model elucidated that MA and EA primarily exert direct toxic effects on tobacco, whereas VA fosters pathogen proliferation, inhibits the enrichment of beneficial bacteria, and synergistically exacerbates the challenges associated with continuous cropping alongside EA. Discussion: These findings suggested discernible disparities in tobacco growth patterns across the various continuous cropping periods. The most pronounced challenges were observed in CC5, whereas CC15 exhibited a substantial reduction in challenges as continuous cropping persisted. VA may play a pivotal role in this phenomenon by interacting with pathogens, beneficial bacterial genera, and EA.

Netrin-1 directs vascular patterning and maturity in the developing kidney.

Blood filtering by the kidney requires the establishment of an intricate vascular system that works to support body fluid and organ homeostasis. Despite these critical roles, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin-1 (Ntn1) is a secreted ligand critical for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by stromal progenitors in the developing kidney, and conditional deletion of Ntn1 from Foxd1+ stromal progenitors (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys that display extended nephrogenesis. Despite expression of the netrin-1 receptor Unc5c in the adjacent nephron progenitor niche, Unc5c knockout kidneys develop normally. The netrin-1 receptor Unc5b is expressed by embryonic kidney endothelium and therefore we interrogated the vascular networks of Foxd1GC/+;Ntn1fl/fl kidneys. Wholemount, 3D analyses revealed the loss of a predictable vascular pattern in mutant kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization in these mutants. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of these results, whole kidney RNA-seq showed upregulation of angiogenic programs and downregulation of muscle-related programs which included smooth muscle-associated genes. Together, our findings highlight the significance of netrin-1 to proper vascularization and kidney development.

Phosphorylation of G protein-coupled receptor kinase 1 (GRK1) is regulated by light but independent of phototransduction in rod photoreceptors.

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser(21) in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark- and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser(21). Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin α-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.